Oligos should be the maximum length because this will help with PCR cleanup and ligation efficiency

Make sure you have some spacer sequence around the restriction site. NEB has a list of the length of the spacer sequence required for each restriction enzyme. (8bp is usually a safe bet)

Order the lowest concentration allowable for the size oligo you want – this will be 50nmole for the 100bp oligo. This will already be more than you’ll need.

If you don’t mind spending more money you can order special “doped” oligo pools where instead of even concentrations of A/T or A/T/C/G or A/T/C, you get 90%A/2%C/8%G, etc. This allows for you to generate a library which is much more likely to produce productive clones.

Double strand the library with modified PCR

Total library DNA should be <25pmol per 100uL reaction

You want to start with 10X the final desired amount of library for PCR

Split into separate 100uL reactions if necessary

Reaction Mix (100uL)

Use the following reaction mix for each PCR reaction:

10 μl 10x Thermo polymerase buffer

10 μl 10x dNTPs (10x = 2.5 mM each dNTP)

5 μl 10 μM FWD primer

5 μl 10 μM REV primer

1 μl Polymerase (taq or vent)

66.5 μl H2O

2.5 μl 10μM library stock

PCR protocol

95 C for 2.5 minutes

Cycle 5 times:

55 C (or whatever temperature is appropriate) for 30 s (annealing)

72 C for 1.5 minutes (elongation)

72 C for 10 minutes (final elongation)

4 C forever

PCR cleanup on the double-stranded libraries

This concentrates the samples and allows for the buffer to be switched to something more appropriate.

PCR purification columns can handle up to 10ug of DNA (100pmol of a 100bp oligo is about 3ug)

Expected recovery from a PCR purification reaction is 90% (from the Invitrogen package)

Alternatively, you can run a sample of the first PCR reaction out on a gel for analysis against a sample of the original library (double stranded should run slightly faster than single stranded), then perform the digest. Doing a PCR cleanup on the digest will remove the cut ends, since they are small.

Transform into compotent cells

Notes

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Expected max library concentration is 10^8 molecules (this is a limit set by the transformation efficiency.) So for step 2, you would like to have 10^9 molecules for a single library transformation (more can be used so a stock can be kept.)
We will typically want a library to have approximately 10^10 molecules (~0.1pmol)